Reactive oxygen species (ROS) are involved in numerous cell-signaling pathways. They have been implicated in over 150 diseases including cancer, arthritis, Parkinson's disease, diabetes, myocardial infarction and atherosclerosis. The need to understand the role of ROS in these processes is evidenced by the 80,000 publications in this field during the last five years alone. Recently, in a paper James D. Watson called among my most important work since the double helix,, he posited a theory that links cancer progression in late stages of the disease to the presence of antioxidants and the role of ROS (Watson, J. Open Biol. 2013, 3, 120144). Despite the immense interest and importance of ROS research, there are no probes that combine reliability, sensitivity, and efficiency for detecting ROS in cell culture, ex vivo, and in vivo. No current commercially available probe can image ROS in vivo, which presents a significant impediment in understanding the role of ROS in their native environment (7). Furthermore, there is no probe which can quantitatively image ROS in cell culture or in vivo. A new class of ROS probes, hydrocyanines, has recently been developed and they hold promise of greatly advancing the study of ROS in vitro, ex vivo and in vivo. The principal investigator of this proposal is the original co-inventor of hydrocyanine probes. In orde to enable unprecedented investigations of cellular processes and disease biology, we propose to develop a commercial family of fluorescent hydrocyanine probes, which accurately detects ROS at low nanomolar concentrations in living cells, tissue samples, blood, and for the first time in whole animals. The objective of this project is to develop novel strategies for the hydrocyanines to establish them as highly accurate and robust ROS probes for in vitro and in vivo research applications. For the first time, quantitative imaging of ROS would also be enabled by the development of ratiometric hydrocyanines probes. The specific aims of this project are: Specific Aim I: Develop novel water-soluble hydrocyanines with low background fluorescence Specific Aim II: Validate the novel hydrocyanines in cell-culture Specific Aim III: Develop ratiometric hydrocyanine probes for quantitative ROS imaging Specific Aim IV: Validate ratiometric hydrocyanines in cell-culture The successful completion of this Phase I SBIR project will establish the commercial feasibility of the low background hydrocyanines, and enable LI-COR to provide biomedical researchers with a robust, simple and versatile toolbox to study ROS in any biological sample. In Phase II, hydrocyanine probes will be developed for industrial drug screening and potential clinical diagnostic applications. Ratiometric hydrocyanines for quantitative in vivo imaging of ROS will also be developed in Phase II. We believe that this proposal is ideally suited for the funding opportunity titled Lab to Marketplace: Tools for Biomedical and Behavioral Research (SBIR [R43/R44]).